Gel electrophoresis, including polyacrylamide gel electrophoresis (PAGE), is a common laboratory technique for estimating the molecular weight of proteins and polypeptides. Gel electrophoresis is used to separate proteins based on their charge, size and/or molecular weight. Under non-denaturing conditions, proteins can be separated based on their charge-to-mass ratio, and the electrophoretic mobility is influenced by the size and shape of the native protein. Under denaturing conditions, for example, in the presence of an anionic detergent such as sodium dodecyl sulphate (SDS), proteins can be separated based on their molecular weights. The anionic detergent both unfolds the protein and provides a uniform negative charge density such that the electrophoretic mobility of the protein is a linear function of the logarithm of the molecular weight.
Methods for quantifying the amount of a protein in a sample are known in the art, including the Bradford assay. The Bradford assay relies on a standard curve generated from known protein standards to determine the total amount of the unknown protein. However, the Bradford assay is typically performed using solutions containing the protein of interest. Purified protein quantity can be measured using spectrophotometric methods if an extinction coefficient is known for the protein of interest. The quantity of a protein can also be determined by mass spectrometry coupled with isotope-labeling methods (e.g., ICAT, iTRAQ, and Tandem Mass Tags). However, spectrophotometric methods are limited to measurements of pure protein in solution. Current methods for quantifying a protein on SDS-PAGE gels involve generating a standard curve from a dilution series of known masses of a purified protein, where each dilution is subjected to electrophoresis in a separate lane of the gel in parallel with the sample containing the target protein of interest.
The present disclosure describes a protein quantitation standard that is useful for quantifying the amount of a protein in an electrophoresis gel, and methods for determining the quantity of a protein in a gel that do not require generating a dilution series of a known protein mass or preparing purified samples of the protein of interest.